Among bacteriophages which infect Clostridium bacteria as obligatory anaerobes, the bacteriophage which was separated from Clostridium tetani by Cowels in 1934 was first known. Since then, bacteriophages capable of lysing various Clostridium bacteria have been reported. There are however rather few reports on such bacteriophages which lyse butyric-acid bacteria. It has been reported by Tahara et al. and Hongo et al. that one species of butyric-acid bacteria is lysed by the enzymes induced by phages HM3 and HM7 which infect Clostridium saccharoperbutylacetonicum. On the other hand, Schlechte described in 1980 such phage 5 which lyses oncolytic Clostridium oncolyticum M55 and Clostridium butyricum H8 {Schlechte, H. et al. "Archiv fur Geschuwulstforschung", 50, 53-57 (1980)}.
The present inventors discovered the new bacteriophage KM1 which is totally different from these reported phages. Starting from this discovery, the present inventors further have found out a special butyric-acid bacterium, namely Clostridium butyricum MII588-Sens 1 strain having a sensitivity to the bacteriophage KM1 from amongst Clostridium butyricum MII588 strains, as well as a mutant, Clostridium butyricum MII588-Res 1 strain which is derived from the Clostridium butyricum MII588-Sens 1 strain and having gained a phage resistance. It has also been found by the present inventors that these strains, i.e., the Sens 1 and Res 1 strains exhibit, both in vitro and in vivo, a strong and stable antagonism against the causative bacteria of acute gastritis and enterocolitis, such as infectious bacteria of food-poisonings and enteric infections, enteropathogenic Escherichia coli and multiple-drug-resistant Shigella strains. Based on these findings, we have found that cells or endospores of the MII588-Sens 1 strain or the MII588-Res 1 strain are effective as an intestinal antiseptic or as a bacteriostatic agent against the causative bacteria of food-poisonings.
Bacteriophage KM1 was isolated from soil. Isolation, purification and biological characteristics of the bacteriophage KM1 have recently been reported in detail by Maeds, Ishii, Tanaka, Mikami and Arai {"J. General Microbiology", 132, 2271-2275 (1986)}.
The characteristics of this phage are as follows.
Shape and host range:
A regular plaque (lysed lesion) of bacteriophage KM1 is transparent and reaches 1-2 mm in diameter 24 hours after incubation. Under electron microscope using the negative staining method, bacteriophage KM1 is in the form of a tadpole and has a hexagonal head whose diameter is 60 nm. A tail having a contractile sheath has a length of 90 nm. Phage 5 of Schlechte et al., which was used for the sake of comparison, has an elliptical head whose diameter is 50.times.80 nm. Its tail is as long as 125 nm, has no sheath and is flexible. The G+C contents were 30.5 mol % for phage KM1 and 35.4 mol % for phage 5, which are both greater than the G+C contents (27-28 mol %) of standard C. butyricum. These two bacteriophages are clearly different from each other, because it is shown that when their DNAs are treated with restriction enzymes Bgl II, Eco RI and Hind III and the resultant DNA fragments are compared by an agarose gel electrophoresis, revealing that their electrophoresis patterns are entirely different. The DNA of phage 5 cannot be digested by Bgl II.
Bacteriophage KM1 cannot lyse entirely any of the butyric-acid bacteria which have been obtained from American Type Culture Collection (ATCC) of U.S.A., Institute of Fermentation, Osaka, and Institute of Applied Microbiology, University of Tokyo, etc. as well as C. butyricum H8 and oncolytic C. oncolyticum M55 (C. butyricum ATCC 13732) which have been supplied by Dr. Schlechte. On the other hand, phage 5 lysed C. butyricum IAM 19004, C. butyricum H8 and oncolytic C. oncolyticum M55.
C. butyricum MII588-Sens 1 and MII588-Res 1 strains are novel microorganisms isolated by the present inventors, and principal morphological and biochemical properties of these two strains may be summarized as shown in the following Tables 1 and 2, in comparison with those of a standard strain, namely, C. butyricum IAM 19001 strain which is stored in Institute of Applied Microbiology, University of Tokyo.
TABLE 1 __________________________________________________________________________ Comparison in morphological properties between Clostridium butyricum MII5588-Sens 1 and Res 1 strains, and Clostridium butyricum IAM 19001 MII588-Sens 1 MII588-Res 1 IAM 19001 __________________________________________________________________________ Shape Gram positive. Gram positive. Gram positive. Straight or slightly Straight or slightly Straight or slightly curved rod, and curved rod, and curved rod, and round at both ends. round at both ends. round at both ends. Size: 0.7-1.4 .times. Size: 0.6-1.4 .times. Size: 0.9-1.6 .times. 3.0-6.5 .mu.m. 2.8-7.0 .mu.m. 5.0-12.5 .mu.m. Normally occurs as Occurs as discrete Occurs as discrete discrete cells. cells, in pair cells, in pairs and/or short chains. and/or short chains. Motility ++ ++ ++ Flangella Peritrichal Peritrichal Peritrichal Endospores Egg-shaped. Egg-shaped. Egg-shaped. 0.8-1.1 .times. 1.9-2.4 .mu.m. 1.0-1.1 .times. 1.9-2.3 .mu.m. 1.0-1.2 .times. 2.0-2.9 .mu.m. Subterminal. Subterminal. Subterminal. __________________________________________________________________________
TABLE 2 ______________________________________ Comparison in biochemical properties between Clostridium butyricum MII588-Sens 1 and Res 1 strains, and Clostridium butyricum IAM 19001 Biochemical MII588- MII588- Properties Sens 1 Res 1 IAM 19001 ______________________________________ Sugar fermentations Sucrose + + + Raffinose + + + Lactose + + + Xylose + + - Glucose + + + Maltose + + + Rhamnose - - - Mannose + + + Arabinose + + + Ribose + + - Glycerol + + - Sorbitol - - - Dulcitol - - - Starch + + + Salicin + + + Diaminopimelic acid DL DL DL of Cell wall Litmus milk Acidic, gas Acidic, gas Acidic, gas and and and coagulated coagulated coagulated Sensitivity to ++ - - bacteriophage KM1 Reduction of nitrates - - + Optimum growth 37.degree. C. 37.degree. C. 37.degree. C. temperature ______________________________________
Clostridium butyricum MII588-Sens 1 and Res 1 strains are spore-forming Gram-positive rods and are obligatory anaerobes. According to the Bergy's classification book "Bergy's Manual of Determinative Bacteriology", 8th edition, Williams & Wilkins Co., (1974), therefore, these two strains are thus classified to be comprised by either the genus Clostridium or the genus Desulfotomaculum. Since the present strains, namely, the Sens 1 and Res 1 strains do not reduce a sulfate in a lactate-sulfate medium, it is without question that they belong to the genus Clostridium. Moreover, their various morphological and biochemical properties are in substantial conformity with those of the butyric-acid bacterium Clostridium butyricum standard strain IAM 19001, as shown in the above Tables. IAM 19001 strain is, however, different from the description of "Bergy's Manual of Determinative Bacteriology" in that IAM 19001 strain lacks the ability for fermentation of xylose and ribose, and is also different from the MII588-Sens 1 and Res 1 strains in this respect. Although the IAM 19001 strain is also different from the MII588-Sens 1 and Res 1 strains with respect to their ability for the fermentations of glycerol, this is immaterial because some of the sugar fermentations is described to vary depending on each specific strain. The MII588-Sens 1 strain can be distinguished from the already known butyric-acid bacterium strains in that it is sensitive to bacteriophage KM1 and can be lysed by the latter phage.